Process for making a protease for foodstuffs

ABSTRACT

AN IMPROVED METHOD FOR MAKING PROTIEN-CONTAINING FOODSTUFF IS PROVIDED THROUGH THE TREATMENT OF THE PROTIEN WITH A PROTEASE WHICH ALLOWS A BREAKDOWN OF THE PROTIEN INTO SIMPLIER PERTIDE UNITS. A METHOD FOR OBTAINING THE NOVEL PROTEASE OF THE INVENTION IS PROVIDED BY CULTURING THE MICROORGANISM STREPTOMYCES TANAKASIS SP. 4680.

PROCESS eon MAKING A PROTEASE FOR FOODS'I'UFFS Filed Feb. 22. 1972 1974 TATSURO TANAKA ETAL 2 Sheets-Sheet 1 O O O 8 6 4 (min) Thermal Stability of Protease EQV 33:3

Effect of Tmperofure on Protease Activity FIG; 2

PROCESS FOR MAKING A PROTEASE FOR FOODSTUFFS g 1974 TATSURO TANAKA Filed Feb.

2 Sheets-Sheet 2 :E w:c3 3329 $0291 08mm oo ZOO Fraction humber (iOml/tube) Chromatographic Separation of Protease 'on (SM-Cellulose FIG.- 3

CULTURE BROTH CENTRIFU GAL SEPARATION SPE CIES M m mm T W w H uw W mm N OE w T m m m U UK T 0 U. H U S F T m M Nl||||||||| R E P U S l FILTRATE RESIDUE DIALYSlS ACETONE PRECIPITATE CM-CELLULOSE COLUMN CHROMATOGRAPHY FIG. 4

United States Patent 3,827,939 PROCESS FOR MAKING A PROTEASE FOR FOODSTUFFS Tatsuro Tanaka, Kyoto, Masaki Terada and Mitsumune Takatsu, Osaka, and Shohei Otani, Hyogo, Japan, assignors to Nissin Shokuhin Kaisha, Ltd., Takatsuki, Osaka, Japan Filed Feb. 22, 1972, Ser. No. 228,072 Claims priority, application Japan, June 23, 1970, 45/56,360; Aug. 13, 1970, 45/71,043 Int. Cl. C12d 13/10 US. Cl. 195-66 R 1 Claim ABSTRACT OF THE DISCLOSURE An improved method for making protein-containing foodstuffs is provided through the treatment of the protein with a protease which allows a breakdown of the protein into simpler peptide units. A method for obtaining the novel protease of the invention is provided by culturing the microorganism Streptomyces tanakasis sp. 4680.

BACKGROUND OF THE INVENTION The treatment of foodstuffs containing protein units with an enzyme to reduce the protein units into simpler peptide units has been proposed as a means to provide a more palatable food product, and one which has increased nutritional value. For example, an improve egg noodle has been proposed which has a superior food value as compare to the conventional egg noodle, and which further is resistant to coagulation upon heating and does not suffer from the characteristic egg odor after prolonged heat treatment common to conventional egg noodles. Such egg noodles further require less time for restoration after heat treatments than is the case with conventional egg noodles. The egg noodles are prepared by treating the egg, prior to incorporation with the flour, with a proteolytic enzyme. The nutritional value of the inclusion of protein in various food products is, of course, well recognized. For example, with respect to the inclusion of egg in egg noodles, when egg is added to a wheat product such as spaghetti, macaroni or noodles, a stable carbon-nitrogen balance is provided, as well as an increased efficiency of metastasis due to the vitamins and inorganic salts contained in the egg.

Various proteolytic enzymes have been tested in order to discover a suitable protease for the preparation of protein foodstuffs having simple peptide units. Bacteria and molds have been proposed, however their efficiency has only been in the range of 20 to 40% in the decomposition of the protein. Certain species of Actinomycrete have been proposed, with a decomposition approaching 80 to 90% of the protein.

In addition to the problem of incomplete reduction of the protein by the various proteolytic enzymes which have been compared, a further difficulty results from the fact that such enzymes are often unstable to heat treatment, and are also readily contaminated by other bacteria.

3,827,939 Patented Aug. 6, 1974 Several bacteria for producing protease have been proposed, including Bacillus thermoproteolyticus and Bacillus stearothermophilus. The protease produced from such bacteria suffer from the disadvantage of having a poor rate of decomposition of the protein. Among the Actinomycete, thermophilic Streptomyces No. 1689 and Thermomonospora No. 2517 (Japanese Patent Publication No. 3479/ 1965) have been proposed, but have not been found to obviate all of the drawbacks mentioned above.

It is therefore an object of the invention to provide an improved protease.

Further object of the invention is the provision of improved foodstuffs through the treatment of protein-containing foodstuffs with a highly efiicient protease.

A further object of the invention is the provision of a thremostable protease which is also resistant to deterioration from other bacteria.

SUMMARY OF THE INVENTION An improved protease is provided from cultures of the microorganism Streptomyces tanakasis sp. 4680. The improved protease is thermostable and provides for the efficient breakdown of protein units, particularly providing improved foodstuffs.

DETAILED DESCRIPTION OF THE INVENTION The improved foodstuffs of the invention are provided from the treatment of the foodstuff with the protease of the invention. The foodstuff may be treated with the protease at any time during its manufacture. It has been found particularly advantageous in some instances, such as in the preparation of egg noodles, to treat the protein-portion of the foodstuff with the protease prior to incorporation with the remaining ingredients.

The various characteristics of the protease of the invention are illustrated with reference to the drawings, where FIG. 1 shows the thermal stability of the protease in the presence of mole Ca-acetate;

FIG. 2 shows the effect of temperature on protease activity at pH 8.0 and 11.0;

FIG. 3 shows the pattern which is obtained by CM-Cellulose column chromatography;

FIG. 4 shows a method of purification of the protease.

The protease of the invention is produced from Streptomyces tanakasis sp. 4680, of mesophilic Actinomycete, for which the characteristics are set forth below:

Major bacteriological properties of the Streptomyces sp. 4680 of mesophilic Actinomycete are shown hereunder.

Morphological properties An aerial mycelium has irregular branches, and forms open spirals to become conidio spores. The mycelium is 5-20 in length, about Lu in width, and red-brownish gray in color. The spore is spherical or ellipsoidal, and 0.8-0.9/J- X 1.0-1.2;1. in size.

The vegetative mycelium forms branches of the long hypha, and does not yield any spores.

CULTURAL FEATURES (Observed on ten days after inoculation) Vegetative mycelium Growth Aerial mycelium (reverse Soluble pigment Emersons agar Very good, velvet-like... Brownish gray Black brown Pale black brown. Czapeks agar do Brownish gray spotted Tan A little purplish red brown.

with white. Glucose-Czapeks agar Good .d0 Pale yellow Pale grayish and yellowish green or a little brown.

Glycerol-Czapeks agar do Pale grayish red or brown Grayish yellow-red Black-brown or brown. Glucose-Asparagine agar Scant Broyg nislifitgray spotted Gray Dark grayish yellow.

W1 w e. Calcium malate agar"-.. No growth. Starch agar Good, smooth Light grayish yellow red- Pale yellowish red Pale grayish red with brown. Glucose a ar dn Brownish gray and white. Leghorn color Pale grayish yellow or brownish yellow.

TABLE-Con tinu ed Vegetative mycelium Growth Aerial mycelium (reverse) Soluble pigment Glucose broth Scent, slight growing on N 0.

bottom. Starch-Ammonium agar Scent No Grahyishhxvhite or yellow- No.

is w i e.

Bouillon agar.. Very good Pale grayish red and white. Grayish red Pale black brown. Glycerol-Stareh-Glutamate ag Very good, velvet-like.-. Brownish gray Gray or brown Do. Gelatin stab Scent, liquified strong Gray or white Very pale brown. EggAlbumin agar Scant- BIOWVIEbSlEI gray (poor Very pale yellow No.

glO Shinobus Tyrosine agar Very good, velvet-like.-- Verydpale greenish white Blgck brown or dark Purplish dark red brown.

an gray. rown. Yeast extracts agar Very good Brownish gray and white. Pale grayish yellow or dull Pale black brown or pale Ski lk yellow. brown.

Peptoii solutio Glycerol agar Black brown or brown. Dull red or pale brown Dull red or pale brown.

White No.

gar Potato plug No growth Physiological properties pH stability; i.e. the remaining activity, is more than 90% a a pH 4.511.0 after heat treatment at 30 C. for 2-4 hours sultable temperflture gmwlh' 33 and at pH 5.0-9.5 after treatment at 70 c. for 10 min- Oxygen: Aerobic utes; furthermore it is more than 50% at pH 6.0-7.5 even Nitrate reduction: Positive after treatment at 80 C. for 10 minutes:

Tyrosinase activity: Positive Amylase activity: Positive TABLE 2PH STABILITY Cellulose activity: Positive (weak) 30 C., 24 Hours Utilization of carbon elements: Good utilization for 25 Glycerol, Sucrose, D-glucose, D-mannose, D-inannit, Remammg Actmty (Percent) D-galactose, Lactose, and Dextrin.

When the above-mentioned properties are compared with the data on both Bergeys Manual of Determination of Bacterialogy, seventh Edition (1957) and S. A. Waksmans The Actinomycetes (1967), the Streptomyces tanakasis sp. 4680 is similar to Streptomyces olivochromogenes, Streptmyces flavochromogenes, and Streptmyces diastatochromogenes, but differs in the following 11.0 100 characteristics: 11.6

Streptomyces Streptomyces Streptmnyces di'izstatoflavochromo- Streptmyces tunakasi's sp. 4580 olivochromogenes chromogenes ge-nea Aerial myeeliunn. Open spiral Closed spiral Ca-malate agar-. Aerial myeelium- No growth Gray"- White.

Colony do Colorless Yellow.

Starch agar Aerial myceliurm. Pale grayish yellow red Slight growth, colorless. Gray White.

Soluble pigment Pale grayish red with brown Colorless Colorle Colony Groyish yellow red with brown Colorless Yellow.

Glucose agar Aerial myceliurm. Gray to white Brown to black Gray Soluble pigment-. Paleugrayish yellow or brownish Colorless Brown.

ye ow. Colony Gray to white Colorless Potato plug Aerial mycelimn. N0 growth Soluble pigment Colony Suitable temperature for growth.. 33 C.-35 G- 37" C 35 C 35 C.

Gelatin--.-.- Greyish White Gray Yellow.

In consequence of above comparison, the Srreptomyces 70 C., 10 min. tanakasis sp. 4680 is reasonably thought to be a variety 55 Remaining c y (P of one of three Actinomycete, though it has such differences as mentioned above from the latter.

Properties of a salted out crude enzyme sample of the thermostable protease which is produced by this strain have been investigated. The test enzyme solution contains $5 mole of Ca++ except in special cases. The most suitable pH is 11.0; the active range is pH 5.013.0. The relation between pH and stability is shown in Table 1:

TABLE 1RELATION BETWEEN pH AND ACTIVITY pH: Activity (extinction coefficient) 80 10 i 6.0 011 P Remaining Activity (percent) 7.0 0.38 19 8.0 g? 47 9.0 051 7'8 0.65 35 10.2 030 i 11.2 0.86 Regarding thermostability, the remaining activity is al- 12.5 0.75 most after treatment at 50 C. for one hour, and

70% even after treatment at 70 C. for one hour. This is illustrated in FIG. 1, and Table 3.

TABLE 3.THERMOSTABILITY (Percent of remained activity at pH 6.9)

Regarding the relation between temperature and activity, the maximum activity appears nearly at 80 C., which is about fifteen times the activity at 30 C. This is illustrated in FIG. 2 and Table 4.

TABLE 4-RELA'I'ION BETWEEN TEMPERATURE AND ACTIVITY Temperature C.): Relative activity (percent) 7 One example of the method of purification is shown in FIG. 4 and the pattern obtained by CM-Cellulose column chromatography is shown in FIG. 3, where (A) shows protein and (B) shows protease activity. The column was 3 x 80 cm.; CM-Cellulose was equilibrated with %00 M. acetate buffer (pH 4.7), and a gradient elution was carried out with 0.09 mole sodium chloride in the same buffer. Although it is a single peak in view of a CM- Cellulose column chromatography, enzyme of higher purity can be obtained by varying diversely the purification method. Influence of various enzyme inhibitors is shown in Table 6.

TABLE 6.-INFLUENOE OF INHIBITION BY VARIOUS In Table 6, an aqueous solution of the acetone precipitate without addition of Ca++ was used as enzyme, in which mole of each compound was added, Zn++, Mg++, Fe+++, Ca++, and KCN had almost no inhibitous efiect, but oxidants such as KMnO NBS, etc. were completely inhibitory. This enzyme can provide hydrolysis of protein of 80-90%, and also has a strong action of curdiness.

In consequence of the above fact, the thermostable protease produced by the Actinomycete, Streptomyces tanakasis sp. 4680 is therefore an excellent protease.

A specimen of Streptomyces tanakasis sp. 4680, has been deposited with the United States Department of Agriculture Northern Regional Research Laboratory, at Peoria, 111., United States of America, and has been given the number NRRL 5402; a sample of this microorganism can be obtained from aforementioned Research Laboratory. (The deposit was made with aforementioned Research Laboratory with all restrictions on the availability to the public being irrevocably removed upon the granting of the patent.)

The following nonlimitative examples are provided for the purpose of illustrating the invention.

EXAMPLE I Culture medium 1: Percent Dextrin 2.0 Defatted soybean powder 2.0 Yeast extract 0.2 Potassium phosphate 0.9 Calcium carbonate 0.5

Into ml. of the above culture medium 1 contained in a Sakaguchis flask of .500 ml. in volume, one loopful of the Streptomyces tanakasis sp. 4680, prepared by means of slant culture, is inoculated. The seed culture is obtained after the above inoculated medium 1 by shaking at 30 C. for 96 hours. Then, 80 ml. of the same culture medium 1 is put in a Sakaguchis flask of 500 ml. in volume, to the extent of approximately 2.0% of the seed thus obtained is inoculated therein, and culture with shaking is carried out at 30 C. for 96 hours. This produces the enzyme of about 3,500 units/ml. in the supernatant liquid of the above cultivated broth.

Measurement of the protease activity Half of the enzyme solution is added to 3 ml. of 0.6% casein solution containing mole of buffer solution (pH 11.0), and the mixture is reacted at 30 C. for 10 min. Then, after 3 ml. of 0.11 mole trichloroacetic acid mixture is added thereto, the precipitate is filtered out, and the extinction coefficient of the filtrate at 275 m is measured. One unit is the amount that can separate for one minute the protein which is soluble in the mixture and When a submerged culturing at 30 C. for about 48 hours by 30 1. jar fermentor is carried out in use of 15 l. of the above-mentioned culture medium 2, the Streptomyces tanakasis sp. 4680 produces the protease of about 5,600 units/ml.

EXAMPLE III The protease of Example II was added to 2.2 kilograms of raw egg with an adjusted pH value of 9.0. Stirring continued for 4 hours at 65 C. to effect a hydrolytic reaction. After hydrolysis, the enzyme was inactivated by boiling for 30 minutes at 100 C., and the pH value was readiusted to 7.0. Spaghetti dough was prepared by mixing 10 kg. of semolina, 2.2 kg. of-the treated egg, and 2.25 liters of water. The resultant spaghetti dough was molded and dried by an ordinary spaghetti-making machine.

What is claimed is:

1. A method of producing an improved protease which comprises preparing a culture medium of Streptomyces tanakasis sp. 4680 under conditions facilitating production of said protease, and isolating said protease from said culture medium, said protease having a high degree of thermostability and being capable of reducing protein into simpler peptide units.

References Cited UNITED STATES PATENTS 3,579,454 5/1971 Collier -66 R X LIONEL M. SHAPIRO, Primary Examiner 

